The authors gratefully acknowledge funding support from the Programa de Pesquisas para o SUS, PPSUS/2020, Minist&#233;rio da Sa&#250;de do Brasil; Funda&#231;&#227;o de Amparo &#224; Pesquisa e Inova&#231;&#227;o do Estado de Santa Catarina, Grant Number: 2021TR000506; Programa de Pesquisa Universal, Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq) do Minist&#233;rio de Ci&#234;ncia e Tecnologia do Brasil, Grant Number: 420996/2023-0; Programa Institucional de Bolsas de Inicia&#231;&#227;o Cient&#237;fica 2022 - 2024, Grant Number: 120815/2023-0. We also thank the volunteers for providing samples.

This protocol was performed as part of the procedures of a clinical trial conducted by our research group (Brazilian Registry of Clinical Trials, ReBEC RBR-729kr8x), and received Ethics Committee approval (process CAAE n&#186; 41063520.4.0 000.0121) and informed consent were obtained from all participants prior to randomization and sample colletction.
The procedure for reconstituting the milk microbiota is done in three steps:
1. Obtaining mother&#39;s raw milk
NOTE: The procedure for obtaining milk can be found elsewhere10,11.
<ol>
	<li>Before the procedure, obtain informed consent from the mother according to the ethical committee and be sure to explain everything that will be done to the mother, addressing any doubts, concerns, fears, and anxieties about the procedure to keep the mother relaxed.</li>
	<li>Have the mother express milk using an electric breast pump into a sterilized container under the supervision of the nurse in charge of the unit, following the established local hygiene standards of each maternity ward.</li>
	<li>After collection, label the container holding the milk with the mother&#39;s information, date, and time of collection. Keep the mother&#39;s own milk (MOM) at a temperature of -4 &#176;C or lower for up to 12 h.</li>
</ol>
2. Defrosting of pasteurized donor&#39;s milk (PDM)
NOTE: The HMBs have their own stock of PDM, frozen at a temperature below -4 &#176;C for a maximum period of up to 6 months, and the procedure is well described in the literature elsewhere12,13,14.
<ol>
	<li>Thaw raw milk or PDM stored in sterilized glass containers with plastic lids in a water bath at 40 &#176;C, ensuring temperature maintenance and a shorter processing time. 
	NOTE: Do not thaw PDM at room temperature or at refrigeration temperature (inside the refrigerator), as thawing times in these cases are extended, which promotes microbial exposure and growth.</li>
	<li>Remove human milk bottles from the water bath before the milk in the bottle reaches a temperature above 5 &#176;C.</li>
</ol>
3. Inoculation of PDM with MOM for microbiome reconstitution
NOTE: The standard volumetric ratio for the process of inoculating PDM with MOM is 90%: 10% (v/v). The steps of the procedure are as follows:
<ol>
	<li>Instruct the technician to wear a lab coat, mask, cap, and gloves.</li>
	<li>Clean the work surface with a surface cleaning solution following local institutional protocol.</li>
	<li>Arrange the materials to be used and prepare the equipment. Preheat the incubator (water bath) with water to 37 &#176;C.</li>
	<li>Plan the final volume of inoculated milk to be used and select an appropriately sized container. Place the container with fresh raw MOM and the PDM in liquid form on the work surface.</li>
	<li>Collect an aliquot of up to 1 mL of the MOM using sterile equipment for subsequent 16S sequencing and bacterial count. Store the aliquot in a DNase/RNase-free cryotube at -80 &#176;C.</li>
	<li>Transfer the remaining volume, equivalent to 10% of the final desired volume of milk, using a sterile volumetric instrument (e.g. syringe, pipette) to a food-safe container to be immersed in a water bath at 37 &#176;C.</li>
	<li>Collect a 1 mL aliquot of the PDM using sterile equipment for subsequent 16S sequencing and bacterial count. Store the aliquot in a DNase/RNase-free cryotube at -80 &#176;C.</li>
	<li>Transfer an amount of PDM equivalent to 90% of the final desired volume of milk using sterile volumetric instrument (e.g., syringe, pipette) to the container with the MOM from the previous step, now forming the inoculated mother&#39;s milk (IM).</li>
	<li>Seal the container with a lid that prevents any external liquid from contaminating the inside of the bottle and immerse it in the water bath at 37 &#176;C for 4 h to allow the microorganisms inoculated in the milk to multiply.</li>
	<li>After 4 h, remove the container with the fermented IM from the water bath, now called reconstituted milk (RM).</li>
	<li>Reserve an aliquot of up to 1 mL using sterile equipment for subsequent 16S sequencing and bacterial count. Store the aliquot store in a DNase/RNase-free cryotube at -80 &#176;C.</li>
	<li>Transfer the remaining volume to a sanitized container for feeding newborns.</li>
</ol>
4. Validation of the method by bacterial culturing on plates
NOTE: All procedures must be performed under sterile conditions to prevent contamination and ensure the accuracy of the dilutions. Bacterial growth can be estimated using methods described elsewhere9,15.
<ol>
	<li>Prepare serial dilutions of each milk sample (PDM, MOM, IM, RM) in sterile saline at 10-1, 10-2, 10-3, and 10-4 concentrations. Mix each dilution thoroughly to ensure uniform bacterial distribution.</li>
	<li>Pippete and spread 100 &#181;L of each diluted sample onto Man-Rogosa-Sharpe (MRS) and Mannitol Salt Agar (MSA) plates in triplicates, using a sterile Drigalski loop or glass spreader. Make sure that the volume is uniformly spread on the agar plate.</li>
	<li>Incubate MRS agar plates in an anaerobic jar containing to create a low-oxygen environment for lactic acid bacteria growth.</li>
	<li>Incubate Mannitol Salt Agar (MSA) plates aerobically to support the growth of Staphylococcus species.</li>
	<li>Incubate the plates at 37 &#177; 2 &#176;C for 48-72 h.</li>
	<li>Select the dilution with a countable number of bacterial colonies (25-250 colonies) on each type of agar</li>
	<li>Count visible colonies on each of the three replicate plates for the selected dilution.</li>
	<li>Calculate the average colony count from triplicate plates for the selected dilution in both MRS and MSA media.</li>
	<li>Determine the number of colony-forming units (CFU) per mL for each sample using the following formula and convert to a base-10 logarithmic scale (log10) to facilitate interpretation. Proceed with statistical analysis of the results. 
	Number of CFU/mL = Number of colonies &#215; total dilution factor/volume plated in mL</li>
</ol>
5. Milk microbiome analysis
NOTE: The milk microbiome analysis can be performed according to the methods and pipeline described elsewhere16. A screenshot of the R script used in this analysis is presented in Supplemental Figure S1.
<ol>
	<li>Centrifuge 500 &#181;L of the sample for 15 s at 180 &#215; g to sediment cells and particulates.</li>
	<li>Collect 350 &#181;L of the supernatant and use it for DNA extraction.</li>
	<li>Quantify the extracted DNA using a spectrophotometer.</li>
	<li>Amplify the V3 and V4 regions of the 16S ribosomal RNA using 20 ng of DNA and primers 357F/805R (see Table of Materials)17.</li>
	<li>Use magnetic beads to purify the resulting amplicons and quantify barcoded amplicons using a DNA fluorimeter.</li>
	<li>Sequence the amplicons in paired-end 250 bp mode.</li>
	<li>Process the resulting sequences by performing quality trimming and adapter removal18. Use paired-end mode (PE) with Phred33 scoring and set a minimum read length of 200 bp to ensure high-quality reads.</li>
	<li>Import paired-end sequence data into QIIME 219 using a manifest file format and the command: qiime tools import.</li>
	<li>Process the imported sequences with DADA2 for denoising, dereplication, and chimera removal by using the command: qiime dada2 denoise-paired. This step generates high-quality representative sequences and a feature table.</li>
	<li>Filter features and sequences to retain only those with sufficient representation using the commands: qiime feature-table filter-features and qiime feature-table filter-seqs.</li>
	<li>Prepare a reference database by extracting regions amplified by the primers (V3-V4) using: qiime feature-classifier extract-reads.</li>
	<li>Use the VSEARCH algorithm for taxonomic classification with the Silva reference database at a 97% identity threshold by executing the command: 
	qiime feature-classifier classify-consensus-vsearch</li>
	<li>Integrate taxonomic assignments with the feature table using BIOM tools: biom add-metadata.</li>
	<li>Perform statistical analyses as described elsewhere20,21.</li>
</ol>

Restoring Pasteurized Donor Milk Microbiota Using Mother's Own Milk

<ol>
	<li>Szyller, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactive+components+of+human+milk+and+their+impact+on+child's+health+and+development,+Literature+Review.">Bioactive components of human milk and their impact on child's health and development, Literature Review.</a> Nutrients. 16 (10), 1487 (2024).</li><li>Fern&#225;ndez, L., Rodr&#237;guez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+milk+microbiota:+Origin+and+potential+uses.">Human milk microbiota: Origin and potential uses.</a> Nestle Nutr Inst Workshop Ser. 94, 75-85 (2020).</li><li>Fern&#225;ndez, L., Ruiz, L., Jara, J., Orgaz, B., Rodr&#237;guez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+the+preservation,+restoration+and+modulation+of+the+human+milk+microbiota.+Implications+for+human+milk+banks+and+neonatal+intensive+care+units.">Strategies for the preservation, restoration and modulation of the human milk microbiota. Implications for human milk banks and neonatal intensive care units.</a> Front Microbiol. 9, 2676 (2018).</li><li>Meier, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+evidence:+Mothers'+own+milk+is+personalized+medicine+for+very+low+birthweight+infants.">More evidence: Mothers' own milk is personalized medicine for very low birthweight infants.</a> Cell Rep Med. 3 (8), 100710 (2022).</li><li>Fewtrell, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictors+of+expressed+breast+milk+volume+in+mothers+expressing+milk+for+their+preterm+infant.">Predictors of expressed breast milk volume in mothers expressing milk for their preterm infant.</a> Arch Dis Child Fetal Neonatal Ed. 101 (6), F502-F506 (2016).</li><li>Abrams, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+human+milk+for+the+high-risk+infant:+Preparation,+safety,+and+usage+options+in+the+United+States.">Donor human milk for the high-risk infant: Preparation, safety, and usage options in the United States.</a> Pediatrics. 139 (1), e20163440 (2017).</li><li>. Brasil &#233; refer&#234;ncia em doa&#231;&#227;o de leite materno Available from: <a target="_blank" href="https://www.gov.br/pt-br/noticias/saude-e-vigilancia-sanitaria/2020/02/brasil-e-referencia-em-doacao-de-leite-materno">https://www.gov.br/pt-br/noticias/saude-e-vigilancia-sanitaria/2020/02/brasil-e-referencia-em-doacao-de-leite-materno</a> (2024)</li><li>Landers, S., Updegrove, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriological+screening+of+donor+human+milk+before+and+after+holder+pasteurization.">Bacteriological screening of donor human milk before and after holder pasteurization.</a> Breastfeed Med. 5 (3), 117-121 (2010).</li><li>Cacho, N. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Personalization+of+the+microbiota+of+donor+human+milk+with+mother's+own+milk.">Personalization of the microbiota of donor human milk with mother's own milk.</a> Front Microbiol. 8, 1470 (2017).</li><li>. Pumping breast milk Available from: <a target="_blank" href="https://www.cdc.gov/nutrition/infantandtoddlernutrition/breastfeeding/pumping-breast-milk.html">https://www.cdc.gov/nutrition/infantandtoddlernutrition/breastfeeding/pumping-breast-milk.html</a> (2023)</li><li>. Expressing and storing breast milk Available from: <a target="_blank" href="https://www.nhs.uk/conditions/baby/breastfeeding-and-bottle-feeding/breastfeeding/expressing-breast-milk/">https://www.nhs.uk/conditions/baby/breastfeeding-and-bottle-feeding/breastfeeding/expressing-breast-milk/</a> (2020)</li><li>Arslanoglu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+the+establishment+and+operation+of+a+donor+human+milk+bank.">Recommendations for the establishment and operation of a donor human milk bank.</a> Nutr Rev. 81 (Supplement_1), 1-28 (2023).</li><li>Moro, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+donor+human+milk:+Update+and+recommendations+from+the+European+Milk+Bank+Association+(EMBA).">Processing of donor human milk: Update and recommendations from the European Milk Bank Association (EMBA).</a> Front Pediatr. 7, 49 (2019).</li><li>Landers, S., Hartmann, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+human+milk+banking+and+the+emergence+of+milk+sharing.">Donor human milk banking and the emergence of milk sharing.</a> Pediatr Clin North Am. 60 (1), 247-260 (2013).</li><li>Stachelska, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Lactobacillus+delbrueckii+and+Streptococcus+thermophilus+strains+present+in+artisanal+raw+cow+milk+cheese+using+real-time+PCR+and+classic+plate+count+methods.">Identification of Lactobacillus delbrueckii and Streptococcus thermophilus strains present in artisanal raw cow milk cheese using real-time PCR and classic plate count methods.</a> Pol J Microbiol. 66 (4), 491-499 (2017).</li><li>Ruiz, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+approaches+for+the+metataxonomic+analysis+of+the+human+milk+microbiome.">Comparison of two approaches for the metataxonomic analysis of the human milk microbiome.</a> Front Cell Infect Microbiol. 11, 622550 (2021).</li><li>Lopez Leyva, L., Brereton, N. J. B., Koski, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+frontiers+in+human+milk+microbiome+research+and+suggested+primers+for+16S+rRNA+gene+analysis.">Emerging frontiers in human milk microbiome research and suggested primers for 16S rRNA gene analysis.</a> Comp Struct Biotechnol J. 19, 121-133 (2021).</li><li>Bolger, A. M., Lohse, M., Usadel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimmomatic:+A+flexible+trimmer+for+Illumina+sequence+data.">Trimmomatic: A flexible trimmer for Illumina sequence data.</a> Bioinformatics. 30 (15), 2114-2120 (2014).</li><li>Bolyen, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=interactive,+scalable+and+extensible+microbiome+data+science+using+QIIME+2.">interactive, scalable and extensible microbiome data science using QIIME 2.</a> Nat Biotechnol. 37 (8), 852-857 (2019).</li><li>McMurdie, P. J., Holmes, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=phyloseq:+An+R+package+for+reproducible+iInteractive+analysis+and+graphics+of+microbiome+census+data.">phyloseq: An R package for reproducible iInteractive analysis and graphics of microbiome census data.</a> PLoS ONE. 8 (4), e61217 (2013).</li><li>R Core Team. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R:+A+language+and+environment+for+statistical+computing.">R: A language and environment for statistical computing.</a> R Foundation for Statistical Computing. , (2024).</li><li>Torrez Lamberti, M. 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The authors declare that they have no competing interests.

Here we present a protocol to be applied in maternity hospitals associated with a human milk bank, aiming to provide PDM with the reconstitution of the milk microbiota from a specific mother who, for various reasons, cannot provide sufficient milk to her newborn. The protocol is simple and essentially based on two steps. The first involves the application of procedures routinely performed in human milk banks for the proper collection and subsequent pasteurization of donor milk, as well as the collection of the mother's o...

Breast milk is widely recognized as the best source of nutrition for newborns, providing not only essential macro and micronutrients but also a complex array of elements, including various metabolites and components of the immune system, such as antibodies, cytokines, and cells1. Additionally, the beneficial properties in breast milk also arise from a community of commensal microorganisms. All these elements of breast milk play a crucial role in the development of the newborn1. The community of microorganisms, known as the microbiota, is influenced by a combination of factors such as diet, maternal lifestyle, type of delivery, and gestational age, resulting in each milk having a microbiota with particular characteristics2. The mechanisms by which the breast milk microbiota benefits neonatal development range from protecting the enteric surface through colonization by commensal bacteria to promoting immune system maturation through the production of metabolites that interact with immune cells in the intestine3. For this reason, the individualized profile of microorganisms in each mother&#39;s milk makes it a form of personalized medicine for newborns4.
The presence of a healthy microbiota can help protect the newborn against colonization by potentially pathogenic microorganisms by occupying the ecological niche and supporting nutrition, especially for premature infants, who are more vulnerable to these external impacts due to being in a hospital environment and having an immature immune system. Moreover, mothers who give birth prematurely often have difficulty producing milk in sufficient quantities for their newborns. Milk production is typically lower the younger the gestational age5. In these cases, the best option for feeding the newborn is pasteurized donor milk provided by Human Milk Banks (HMB)6.
Brazil has the largest and most extensive network of HMBs in the world7. This network collects, prepares, and distributes more than 160,000 L of pasteurized donor human milk (PDM) throughout the country at no cost to parents, through the national unified health system (Sistema &#218;nico de Sa&#250;de), managed by the Ministry of Health7. Pasteurization is essential to ensure the biosafety of the donated milk intended for newborns. This process is usually carried out using the Holder method, which involves immersing a container of raw donor breast milk in a water bath at 62.5 &#176;C for 30 min6,8. The resulting PDM is then stored under refrigeration until it is ready for consumption. However, pasteurization reduces the viability of the milk&#39;s commensal microbiota and decreases the bioavailability of various bioactive components. This is because the pasteurization process is standardized to prevent the transmission of pathogenic microorganisms to the newborn. In this context, in 2017, Cacho and colleagues described the possibility of reconstituting the commensal microbiota of a mother&#39;s own milk by inoculating it in various proportions into PDM9. Their findings demonstrated that using a 10% concentration of the mother&#39;s own milk (MOM) is the optimal concentration for maintaining the original composition of the MOM microbiome profile. This approach reduces the risk of overgrowth of specific bacteria, which, although typically commensal, could become harmful if disproportionately present in the reconstituted milk. Specifically, the study observed that a 1:10 dilution (10% MOM + 90% PDM), incubated for 4 h, resulted in a well-balanced bacterial load&#160;&#8212;&#160;approximately 60% of that found in undiluted MOM -- indicating a safer and more effective approach. In contrast, higher dilutions, such as 3:10, led to excessive bacterial growth, highlighting the critical need for controlled concentrations and conditions to ensure safety.
In this article, we demonstrate that this procedure of reconstituting the breast milk microbiota in PDM can be achieved in real-world situations within the routine care of a maternity hospital and its associated human milk bank.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Electric breast milk pump</td>
			<td>Horigen</td>
			<td>XN-2219M2</td>
			<td>Soft pump dual plus</td>
		</tr>
		<tr>
			<td>Pyrex&#160;media bottles</td>
			<td>Corning&#160;</td>
			<td>CLS1395100</td>
			<td>Glass containers with plastic lids&#160;</td>
		</tr>
		<tr>
			<td>1000 &#181;L pipette Labmate pro</td>
			<td>Corning HTL SA&#160;&#160;</td>
			<td>5666</td>
			<td>Variable volume pipettor&#160;</td>
		</tr>
		<tr>
			<td>Cryogenic vial&#160;</td>
			<td>Corning&#160;</td>
			<td>CLS431417</td>
			<td>DNase/RNase-free 2 mL vials&#160;</td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Corning&#160;</td>
			<td>CLS431470</td>
			<td>DNase/RNase-free 15 mL tubes&#160;</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>EcoSonics</td>
			<td>Q3.0/40A</td>
			<td></td>
		</tr>
		<tr>
			<td>Man&#8211;Rogosa&#8211;Sharpe (MRS)&#160;</td>
			<td>Difco</td>
			<td>288210</td>
			<td>Lactic acid bacteria growth media</td>
		</tr>
		<tr>
			<td>Mannitol Salt Agar (MSA)</td>
			<td>Himedia</td>
			<td>MH118</td>
			<td>Staphylococcus&#160; growth media</td>
		</tr>
		<tr>
			<td>Anaerobic Jar</td>
			<td>Permution</td>
			<td></td>
			<td>Create a low-oxygen environment for lactic acid bacteria growth</td>
		</tr>
		<tr>
			<td>Extracta&#160; Kit &#8211; DNA e RNA</td>
			<td>Loccus</td>
			<td>MPTA-PV16-B Y</td>
			<td>DNA extraction Kit</td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Promega</td>
			<td>E6150</td>
			<td>Quantus DNA</td>
		</tr>
		<tr>
			<td>GoTaqG2</td>
			<td>Promega</td>
			<td>M7841</td>
			<td>PCR amplication System</td>
		</tr>
		<tr>
			<td>Quantus Fluorometer</td>
			<td>Promega</td>
			<td>E5150</td>
			<td>DNA / RNA quantitation kit</td>
		</tr>
		<tr>
			<td>MiSeq System</td>
			<td>Illumina Inc.</td>
			<td>M-GL-00006 v4.0</td>
			<td>Sequencing equipment</td>
		</tr>
		<tr>
			<td>MiSeq Reagent Kit v2 (300-cycles)</td>
			<td>Illumina Inc.</td>
			<td>MS-102-2002</td>
			<td>Sequencing kits and reagents</td>
		</tr>
		<tr>
			<td>Software name&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trimmomatic</td>
			<td>http://www.usadellab.org/cms/index.php?page=trimmomatic</td>
			<td></td>
			<td>Read trimming tool for Illumina NGS data</td>
		</tr>
		<tr>
			<td>QIIME 2&#160;</td>
			<td>https://docs.qiime2.org/2024.10/</td>
			<td></td>
			<td>Microbiome analysis package&#160;</td>
		</tr>
		<tr>
			<td>Primer name</td>
			<td>Primer sequence</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>16S_357F&#160;</td>
			<td>TCGTCGGCAGCGTCAGA 
			TGTGTATAAGAGACAGC 
			CTACGGGNGGCWGCAG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>16S_805R</td>
			<td>GTCTCGTGGGCTCGGAG 
			ATGTGTATAAGAGACAGG 
			ACTACHVGGGTATCTAATC</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

individualized reconstitution of human milk microbiota: a feasible approach in real-world settings

Mother&#39;s own milk (MOM) is the most complete nutritional resource for newborns. In cases where mothers are unable to produce sufficient milk or cannot breastfeed, the preferred alternative is pasteurized donor human milk (PDM), which is routinely provided by human milk banks. PDM offers a superior range of nutritional and immunological elements compared to any commercially available formula. However, to ensure biosafety, PDM undergoes pasteurization, a process that inactivates commensal microbiota and reduces certain bioactive compounds. This study presents a protocol designed to restore the microbiota of PDM using MOM as a microbial source, adapting the approach to a real-world clinical setting.
The protocol was implemented in a clinical trial conducted at a maternity hospital and its associated human milk bank, with the aim of providing personalized donor milk to preterm infants whose mothers cannot produce sufficient milk. The methodology involves inoculating PDM with 10% of MOM, followed by incubation at 37 &#176;C for 4 h. Microbiological analysis demonstrated successful bacterial growth in the inoculated milk (IM) post incubation, with the microbiota profile of the reconstituted milk (RM) closely resembling that of MOM, indicating effective microbiota restoration. These results suggest that the reconstitution protocol is feasible for implementation in neonatal care, with the potential to enhance the nutritional and immunological quality of PDM, thereby supporting the health and development of non-breastfed newborns.

Bacterial growth analysis by plate culturing 
The microbiological tests conducted to validate the method of inoculating PDM with the MOM, as proposed by Cacho et al.9, demonstrated that, based on the CFU/mL count, there were no significant differences between the inoculated milk samples before and after the 4 h incubation. However, differences in the number of colonies were observed at these two time points, indicating bacterial growth during incubation on MSA for Staphyl...

Individualized Reconstitution of Human Milk Microbiota: A Feasible Approach in Real-World Settings

The article describes a protocol for reconstituting pasteurized donor milk microbiota using the mother's own milk microbiota in practical, real-world settings. It demonstrates effective bacterial growth and microbiome modulation, supporting the feasible application of this procedure within the routine care of a maternity hospital and its associated human milk bank.

Mother&#39;s own milk (MOM) is the most complete nutritional resource for newborns. In cases where mothers are unable to produce sufficient milk or cannot ...

individualized-reconstitution-human-milk-microbiota-feasible-approach

Research

JoVE Journal

Medicine

292 Views.  Federal University of Santa Catarina. The article describes a protocol for reconstituting pasteurized donor milk microbiota using the mother's own milk microbiota in practical, real-world settings. It demonstrates effective bacterial growth and microbiome modulation, supporting the feasible application of this procedure within the routine care of a maternity hospital and its associated human milk bank.

Video: Individualized Reconstitution of Human Milk Microbiota: A Feasible Approach in Real-World Settings

Using Continuous Data Tracking Technology to Study Exercise Adherence in Pulmonary Rehabilitation

Pulmonary rehabilitation (PR) is an important component in the management of respiratory diseases. The effectiveness of PR is dependent upon adherence to exercise training recommendations. The study of exercise adherence is thus a key step towards the optimization of PR programs. To date, mostly indirect measures, such as rates of participation, completion, and attendance, have been used to determine adherence to PR. The purpose of the present protocol is to describe how continuous data tracking technology can be used to measure adherence to a prescribed aerobic training intensity on a second-by-second basis.
In our investigations, adherence has been defined as the percent time spent within a specified target heart rate range. As such, using a combination of hardware and software, heart rate is measured, tracked, and recorded during cycling second-by-second for each participant, for each exercise session. Using statistical software, the data is subsequently extracted and analyzed. The same protocol can be applied to determine adherence to other measures of exercise intensity, such as time spent at a specified wattage, level, or speed on the cycle ergometer. Furthermore, the hardware and software is also available to measure adherence to other modes of training, such as the treadmill, elliptical, stepper, and arm ergometer. The present protocol, therefore, has a vast applicability to directly measure adherence to aerobic exercise.

Pulmonary rehabilitation is widely recognized in the management of respiratory diseases. A key component to successful pulmonary rehabilitation is adherence to the recommended exercise training. The purpose of the present protocol is to describe how continuous data tracking technology can be used to precisely measure adherence to a prescribed aerobic training intensity.

Pulmonary rehabilitation (PR) is an important component in the management of respiratory diseases. The effectiveness of PR is dependent upon adherence to ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and follow-up of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2-&#916;&#916;ct 13. Control values were used as baseline and results are shown in logarithmic scale.

Exosomal miRNA Analysis in Non-small Cell Lung Cancer NSCLC Patients&#039; Plasma Through qPCR: A Feasible Liquid Biopsy Tool

This protocol describes the feasibility to perform miRNA profiling in exosomes, released in plasma of NSCLC patients, through a commercial exosome isolation kit with Proteinase K and RNAse treatments, in order to avoid circulating miRNAs contamination and evaluate their biomarker features in NSCLC.

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

A Novel Approach for the Administration of Medications and Fluids in Emergency Scenarios and Settings

The available routes of administration commonly used for medications and fluids in the acute care setting are generally limited to oral, intravenous, or intraosseous routes, but in many patients, particularly in the emergency or critical care settings, these routes are often unavailable or time-consuming to access. A novel device is now available that offers an easy route for administration of medications or fluids via rectal mucosal absorption (also referred to as proctoclysis in the case of fluid administration and subsequent absorption). Although originally intended for the palliative care market, the utility of this device in the emergency setting has recently been described. Specifically, reports of patients being treated for dehydration, alcohol withdrawal, vomiting, fever, myocardial infarction, hyperthyroidism, and cardiac arrest have shown success with administration of a wide variety of medications or fluids (including water, aspirin, lorazepam, ondansetron, acetaminophen, methimazole, and buspirone). Device placement is straightforward, and based on the observation of expected effects from the medication administrations, absorption is rapid. The rapidity of absorption kinetics are further demonstrated in a recent report of the measurement of phenobarbital pharmacokinetics. We describe here the placement and use of this device, and demonstrate methods of pharmacokinetic measurements of medications administered by this method.

This study presents a novel device that offers an easy route to quickly provide medications and fluids in patients with limited or difficult IV access. This small-diameter device is placed in the distal third of the rectum, allowing for ongoing medication and fluids administration.

The available routes of administration commonly used for medications and fluids in the acute care setting are generally limited to oral, intravenous, or ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

Cardiopulmonary Bypass in a Mouse Model: A Novel Approach

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization and for minimizing organ damage resulting from prolonged extracorporal circulation. The goal of this paper was to demonstrate a fully functional and clinically relevant model of cardiopulmonary bypass in a mouse. We report on the device design, perfusion circuit optimization, and microsurgical techniques. This model is an acute model, which is not compatible with survival due to the need for multiple blood drawings. Because of the range of tools available for mice (e.g., markers, knockouts, etc.), this model will facilitate investigation into the molecular mechanisms of organ damage and the effect of cardiopulmonary bypass in relation to other comorbidities.

This paper describes how to perform cardiopulmonary bypass in mice. This novel model will facilitate the investigation of the molecular mechanisms involved in organ damage.

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization ...

Individualized Stem-positioning in Calcar-guided Short-stem Total Hip Arthroplasty

Bone- and soft-tissue sparing short stems are increasingly used in total hip arthroplasty (THA). However, there are a large variety of models of short stems, differing in design and function. Calcar-guided short stems provide an anatomical curvature in the medial calcar region, thus, positioning is done individually alongside the calcar in the "round-the-corner" technique. Depending on the level of the neck's osteotomy, stems can be aligned individually in a large bandwidth of varus- and valgus anatomies. This differs from conventional total hip arthroplasty and potentially includes a severe learning curve. Given that a great variety of caput-collum-diaphyseal (CCD)-angles can be retained, the reconstruction of femoro-acetabular offsets can be achieved precisely. However, particularly extensive varus- and valgus positioning has raised concerns in regard to stability and bone remodeling. The purpose of the present manuscript is to showcase the implantation technique in calcar-guided short-stem THA and to summarize short-term clinical and radiological results.

This protocol describes the round-the-corner technique and the individualized stem-positioning of calcar-guided short stems alongside the medial calcar, depending on the level of the osteotomy. This differs from conventional total hip arthroplasty and includes a learning curve.

Bone- and soft-tissue sparing short stems are increasingly used in total hip arthroplasty (THA). However, there are a large variety of models of short ...

Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the genotype-directed therapy model currently in use has several limitations. First, rare or understudied mutation groups are excluded from definitive clinical trials. Moreover, as additional modulator drugs enter the market, it will become difficult to optimize the modulator choices for an individual subject. Both of these issues are addressed with the use of patient-derived, individualized preclinical model systems of CFTR function and modulation. Human nasal epithelial cells (HNEs) are an easily accessible source of respiratory tissue for such a model. Herein, we describe the generation of a three-dimensional spheroid model of CFTR function and modulation using primary HNEs. HNEs are isolated from subjects in a minimally invasive fashion, expanded in conditional reprogramming conditions, and seeded into the spheroid culture. Within 2 weeks of seeding, spheroid cultures generate HNE spheroids that can be stimulated with 3',5'-cyclic adenosine monophosphate (cAMP)-generating agonists to activate CFTR function. Spheroid swelling is then quantified as a proxy of CFTR activity. HNE spheroids capitalize on the minimally invasive, yet respiratory origin of nasal cells to generate an accessible, personalized model relevant to an epithelium reflecting disease morbidity and mortality. Compared to the air-liquid interface HNE cultures, spheroids are relatively quick to mature, which reduces the overall contamination rate. In its current form, the model is limited by low throughput, though this is offset by the relative ease of tissue acquisition. HNE spheroids can be used to reliably quantify and characterize CFTR activity at the individual level. An ongoing study to tie this quantification to in vivo drug response will determine if HNE spheroids are a true preclinical predictor of patient response to CFTR modulation.

Here we describe a method to generate three-dimensional spheroid cultures of human nasal epithelial cells. Spheroids are then stimulated to drive Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-dependent ion and fluid secretion, quantifying the change in the spheroid luminal size as a proxy for CFTR function.

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the ...

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...